CN116479008A - 一种调控大豆根瘤数目的基因及其应用 - Google Patents
一种调控大豆根瘤数目的基因及其应用 Download PDFInfo
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Abstract
本发明属于植物基因工程技术领域，尤其涉及一种调控大豆根瘤数目的基因及其应用。该基因为大豆AP2转录因子GmWRI1c，本发明公开了大豆AP2转录因子GmWRI1c及其核苷酸序列、编码的氨基酸和引物对，提供了通过该GmWRI1c基因或含有该GmWRI1c基因的表达载体或宿主细胞在促进大豆根瘤发育和/或增加根瘤数目上的应用，为提高大豆产量与品质的生产提供了新的基因资源和手段，具有重要意义。
Description
技术领域
本发明属于本发明属于植物基因工程技术领域，尤其涉及一种调控大豆根瘤数目的基因及其应用。
技术背景
根瘤是豆科植物中一个独特的结构。根瘤菌将空气中的氮固定为氨，从而为其宿主植物的生长和发育提供氮，宿主的根和根瘤为根瘤菌提供碳和营养物质(Mendoza etal.,2005)。
共生结瘤是一个复杂的性状，受结瘤因子、内源激素和外部环境的影响。以前的研究发现，结瘤因子直接调节豆科植物的结瘤。结瘤因子包括结瘤受体(NINs)、结瘤因子受体(NFR1s)、共生受体激酶(SYMRK/DMI2)、钙调蛋白激酶(CCaMK/DMI3)、结瘤信号通道蛋白(NSP)和结瘤标记基因(ENOD40)(Mu et al.,2013；Roscoe et al.,2015)。植物激素在结瘤过程中也起着重要作用(Lotan et al.,1998)，生长素和细胞分裂素促进结瘤(Jinye etal.,2008；et al.,2011；Feeney et al.,2013)，而脱落酸抑制结瘤(Baudet al.,2009；Hyun et al.,2014)。豆科植物的结瘤同样受环境因素的影响，例如，光照、氮和盐胁迫会抑制豆科植物的结瘤(Suzuki et al.,1997；Tsuchiya et al.,2004；Keith etal.,1994)。
现有研究中，专利CN109053869A公开了大豆核孔蛋白基因GmNup96在调节植物根瘤发育中的应用，专利CN110004157A公开了一种通过GmSGF14H和GmSGF14I基因增加根瘤数目和促进根瘤发育的转基因植物培育方法，专利CN114574518A公开了利用GmNAC181基因促进豆科作物结瘤尤其是耐盐结瘤的方法。
也有研究表明，脂质代谢基因参与调控大豆的结瘤(Baud et al.,2009；Kroj etal.,2003；Yamamoto et al.,2010)，但仍不清楚脂肪酸如何影响大豆结瘤的数量。研究脂质代谢与结瘤之间的相互作用，确定参与大豆结瘤的脂质代谢基因以及这一过程的调控，对于培育新的、高品质的大豆品种具有重要意义。
WRI1属于AP2转录因子超级基因家族的ANT类群，通过与其靶基因启动子中保守的AW-box结合，促进脂肪酸的合成(Nikolau et al.,2003；Guchhait etal.,1974)。WRI1首次在拟南芥皱纹种皮突变体中被发现(Konishi et al.,1994)。已有研究表明，AtWRI1及其同源物BnWRI1、HaWRI1、CiWRI1和GmWRI1a可以显著提高不同植物种子的含油量(Salie etal.,2016；Thelen et al.,2002；张芮等.,2018；Yang et al.,2016；Aslan et al.,2015；Abbadi et al.,2000)。在大豆中，GmWRI1b可以在田间条件下改善植物结构并增加产量(Choi et al.,2000)。
目前尚未有关于GmWRI1在调控大豆根瘤数目和影响根瘤发育的相关研究报道。
发明内容
为了解决现有技术中的问题，本发明的目的之一在于提供一种调控大豆根瘤数目的基因，该基因为大豆AP2转录因子GmWRI1c，其核苷酸序列如SEQ ID NO:1所示，编码的蛋白质氨基酸序列如SEQ ID NO:2所示。
本发明还提供一种含有上述的大豆AP2转录因子GmWRI1c的表达载体。该表达载体为pCAMBIA1305-GmWRI1c-GFP表达载体，其以大豆cDNA为模板，经PCR反应后得到的PCR产物连接pCAMBIA1305-GFP载体得到。
优选的，所述PCR反应使用的上游引物序列如SEQ ID NO.3所示，下游引物如SEQID NO.4所示。
本发明还提供一种含有上述大豆AP2转录因子GmWRI1c的宿主细胞，所述宿主细胞为发根农杆菌K599。
本发明还提供上述的大豆AP2转录因子GmWRI1c和/或表达载体和/或宿主细胞在促进大豆根瘤发育和/或增加根瘤数目上的应用。
本发明的有益效果在于：
1)本发明研究发现GmWRI1c基因能够调控大豆根瘤数量，通过在植株中过表达GmWRI1c，能够提高转基因植株的根瘤发育，提高毛状根根瘤数量，以及促进糖酵解和脂肪酸从头合成基因的表达，增加根与根瘤中棕榈酸(C16:0)的比例。
2)本发明利用生物化学与分子生物学以及转基因技术手段探讨GmWRI1c基因调控大豆结瘤的分子机理，为提高大豆产量与品质的生产提供了新的基因资源，具有重要意义。
附图说明
图1为本发明实施例中转基因根系GmWRI1c-OE鉴定，其中a为转基因毛状根的鉴定，b为转基因根瘤的鉴定；
图2为本发明实施例中转基因根系的总脂肪酸含量，其中a为毛状根总脂肪酸含量，b为根瘤总脂肪酸含量；
图3为本发明实施例中转基因根系的脂肪酸组成，其中a为毛状根中脂肪酸组成，b为根瘤中脂肪酸组成；
图4为本发明实施例中转基因根系的的糖酵解和从头脂肪酸合成相关基因的转录水平，其中a为毛状根的转录水平，b为根瘤的转录水平；
图5为本发明实施例中转基因大豆根中GmWRI1c的转录水平；
图6为GmWRI1c-OE转基因大豆毛状根的根瘤数目与对照组相比(n＝5)，过表达GmWRI1c能够极显著(P<0.01)增加根瘤数目。
上述图1-6中数值均为三个技术重复的平均值±SD；通过t检验进行显著性分析，*P<0.05和**P<0.01。
具体实施方式
为了便于理解，下面结合实施例对本发明的技术方案做出更为具体的说明：
实施例1
大豆AP2转录因子GmWRI1c的获得
使用在拟南芥、苜蓿和油菜中已被鉴定基因功能的WRI基因(AtWRI1、AtWRI2、AtWRI3、AtWRI4、MtWRI1和BnWRI1)进行进化分析并构建进化树，发现6个与AtWRI1高同源性的基因，分别命名为GmWRI1a、GmWRI1b、GmWRI1c、GmWRI1d、GmWRI1e、GmWRI1f。利用phytozome数据库对6个GmWRI1s的组织表达模式进行分析，发现GmWRI1c在根瘤中特异性表达。GmWRI1c核苷酸序列如SEQ ID NO:1所示，其编码的氨基酸序列如SEQ ID NO:2所示。
实施例2
(1)pCAMBIA1305-GmWRI1c-GFP表达载体构建：
利用1对引物GmWRI1c.XbaI.F(SEQ ID NO:3)和GmWRI1c.BamHI.R(SEQ ID NO:4)，从大豆天隆1号cDNA进行PCR扩增(表1)并酶切pCAMBIA1305空载(表2)，采用同源重组的方法将目的片段即GmWRI1c与酶切后的空载连接(表3)，得到连接产物即pCAMBIA1305-GmWRI1c-GFP表达载体。
表1PCR扩增体系
PCR反应程序：98℃5min；98℃10s，58℃15s，68℃20s，共36个循环；68℃5min。
表2酶切体系
酶切反应程序：37℃4h；80℃15min，4℃∞。
表3连接反应体系
酶切反应程序：37℃4h；80℃15min，4℃∞。
(2)宿主细胞构建：
2.1重组载体转化大肠杆菌：向50μl刚融化的大肠杆菌感受态细胞(Trans1-T1)中加入5μl上述制备的连接产物；轻轻地拨动管底混匀之后立即插入冰上静置冰浴25min；小心取出后轻轻地放入42℃恒温水浴锅中水90s；然后立即静置于冰上冰浴5min；在无菌操作台中，向管中的转化菌液加入700μl不含抗生素的LB液体培养基；最后将含有菌液的离心管置于37℃恒温摇床进行孵育1h左右(180r/min)。取100μl孵育后的菌液用无菌涂布器涂布于含有卡那霉素(Kana)的LB固体平板上，晾干后将平板倒放于37℃恒温培养箱中进行过夜培养(约12-14h)；
2.2重组载体阳性克隆筛选：挑取单个菌落于含有600μl LB液体培养基(含Kana)的2.0mL无菌圆底离心管中，置于37℃恒温摇床培养4～5h(220r/min)。采用表1的PCR扩增方法进行菌液PCR，根据琼脂糖凝胶电泳检测结果，挑取经初步判断可能导入目的基因的菌液进行DNA测序，确认GmWRI1c CDS序列；
2.3重组载体转化农杆菌：将测序正确的单克隆菌液加入含有5mL LB液体培养基(含Kana)的15mL摇菌管中，置于37℃恒温摇床培养12h(220r/min)。使用Axgeny试剂盒提取质粒。将保存在-80℃冰箱中的农杆菌感受态细胞(K599)取出，在室温条件下让其慢慢融化，当菌液刚刚融化为液体状态时立即插入冰盒内。在无菌操作台内用移液枪向每50μl感受态细胞中加入0.2μg的质粒，轻轻地拨动管底混匀之后，然后依次置于冰上静置5min、液氮冰冻5min、37℃水浴加热5min、冰上静置5min。在无菌操作台中用1000μl移液枪吸取700μl恢复室温且无任何抗生素的YEP培养液加入到上述含有转化菌液的离心管中，然后将菌液置于28℃摇床220r/min恒温培养2h。培养结束后，吸取100μl菌液用无菌涂布器涂布于YEP固体培养基上(YEP培养基含有卡那和利福平抗生素)，倒置放在28℃培养箱中恒温培养2～3d。待菌落长至2～3mm时，在无菌操作台中挑取单个菌落于含有600μlYEP液体培养基(含对应抗生素)的2.0mL无菌圆底离心管中，置于28℃恒温摇床培养20～24h(220r/min)。菌液PCR正确的条带的菌液即为所需宿主细胞，菌液与甘油等比例混合置于-80℃冰箱保存用于后续实验。
实施例3
大豆过表达GmWRI1c转基因植株的获得及其对大豆植株的影响
选取饱满的天隆一号种子，用hpe对种子消毒，在24～26℃、16h光照/天的条件下使其萌发一周。
转化过程如下：
(1)挑取实施例2中含pCAMBIA1305-GmWRI1c-GFP的宿主细胞，接种到1ml TY液体培养基中(链霉素50μg/ml、卡那霉素50μg/ml)于恒温摇床上28℃，180rpm培养过夜，然后取200ul涂于TY固体培养基(链霉素50μg/ml、卡那霉素50μg/ml)，过夜培养28度，对照为空载。
(2)选取健康的萌发幼苗，收集步骤(1)中的菌体注射至大豆子叶下胚轴。
(3)将注射后的大豆幼苗扎根转移到蛭石中，2周后剪去主根并移栽到营养土中，在大豆R5时期取转基因根与根瘤，置于-80℃冰箱保存。
用qRT-PCR鉴定出转基因根系(GmWRI1c-OE)，结果如图1所示，图中a为转基因毛状根的鉴定，共鉴定3个独立的转基因根系且相对表达量显著高于野生型。b为转基因根瘤的鉴定并分析，共鉴定3个独立的转基因根瘤且相对表达量显著高于野生型。分析GmWRI1c-OE转基因根和根瘤的总脂肪酸含量与脂肪酸组分，结果如图2所示，与野生型(WT)相比，过表达GmWRI1c的株系根和根瘤中总脂肪酸含量分别增加了38.22％和32.8％。
如图3所示，过表达GmWRI1c会特异性提高棕榈酸的比例。图中a显示与野生型相比，3个独立的转基因毛状根中的棕榈酸比例分别提高8.46％、8.16％、10.40％，同样在根瘤中也有相同的趋势，图中b显示3个独立的转基因根瘤中的棕榈酸比例分别提高7.58％、7.58％、6.62％，其他四种脂肪酸组分无明显差异。
综上所述，GmWRI1c对脂肪酸的合成有正向调控的作用，尤其是对棕榈酸的合成具有促进作用，表明GmWRI1c促进了脂质代谢途径，最终对大豆根瘤数量的增加造成影响。
为进一步验证上述推论，继续对过表达转基因毛状根与根瘤中参与质体糖酵解和脂肪酸从头合成基因的表达量进行分析。发现过表达GmWRI1c可以极显著(P≤0.01)提高质体糖酵解基因(GmPDHE、GmPKP和GmDLD等)和脂肪酸从头合成基因(GmPDCT、GmACP和GmMCAT等)的表达水平，结果参见图4。糖酵解为脂肪酸从头合成提供乙酰辅酶A(Co-A)底物，而脂肪酸从头合成的主要产物是棕榈酸。以上结果表明，GmWRI1c通过调节参与糖酵解和脂肪从头生成的相关基因，特异性地增加了大豆中棕榈酸的含量。
如图5所示，使用根瘤菌侵染大豆根部后，GmWRI1c的表达量在根瘤菌侵染6小时后极显著(P≤0.01)上调。
统计转基因大豆毛状根的根瘤数量，如图6所示，过表达GmWRI1c能够极显著(P<0.01)增加根瘤数目，对照毛状根平均产生43.4个根瘤(n＝5)，转基因毛状根平均产生69.8个根瘤(n＝5)，增幅达到60.83％。表明过表达GmWRI1c可以通过增加大豆根瘤数目的方式促进大豆结瘤。
以上实施方式仅用以说明本发明的技术方案，而并非对本发明的限制；尽管参照前述实施方式对本发明进行了详细的说明，本领域的普通技术人员应当理解：凡在本发明创造的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明创造的保护范围之内。
Claims (10)
1.一种调控大豆根瘤数目的基因，其特征在于，该基因为大豆AP2转录因子GmWRI1c，其核苷酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的一种调控大豆根瘤数目的基因，其特征在于，GmWRI1c基因编码的蛋白质氨基酸序列如SEQ ID NO:2所示。
3.如权利要求1所述的一种调控大豆根瘤数目的基因在促进大豆根瘤发育和/或增加根瘤数目上的应用。
4.一种含有如权利要求1所述的大豆AP2转录因子GmWRI1c的表达载体。
5.如权利要求4所述的表达载体，其特征在于，该表达载体为pCAMBIA1305-GmWRI1c-GFP表达载体，其以大豆cDNA为模板，经PCR反应后得到的PCR产物连接pCAMBIA1305-GFP载体得到。
6.如权利要求5所述的表达载体，其特征在于，所述PCR反应使用的上游引物序列如SEQID NO.3所示，下游引物如SEQ ID NO.4所示。
7.如权利要求4或5所述的表达载体在促进大豆根瘤发育和/或增加根瘤数目上的应用。
8.一种宿主细胞，其特征在于，含有如权利要求1所述的大豆AP2转录因子GmWRI1c，或含有如权利要求5所述的表达载体。
9.如权利要求8所述的宿主细胞，其特征在于，所述宿主细胞为发根农杆菌K599。
10.如权利要求8所述的宿主细胞在促进大豆根瘤发育和/或增加根瘤数目上的应用。
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